Device and method for reducing the permeability of the cornea

ABSTRACT

The present invention provides a device and a method for non-invasively enhancing the integrity of the cornea by applying a magnetic field, thereby treating eye disorders.

FIELD OF THE INVENTION

The present invention relates to treating eye conditions by reducing thecorneal permeability by alternating magnetic field.

BACKGROUND OF THE INVENTION

The corneal epithelium functions is the principal barrier to thepenetration of noxious substances into the anterior chamber, and assistsin protecting the cornea by maintaining normal hydration and retainingocular surface integrity. This diffusion barrier blocks the penetrationof polarized substances such as water or ions as well as macromoleculesand cells, and represents 50% of the total diffusion barrier of thehealthy cornea. The corneal epithelium consists of five cell layers ofstratified squamous nonkeratinized cells and an underlying basal layer.The barrier function depends on epithelial cell tight junctions, theassembly of which is regulated by intra and extra-cellular calcium. Evenminor lesions of the corneal surface, too small to be recognized in thedaily clinical setting, may result in impairment of the cornealepithelial barrier function that can be quantified in vivo by means ofobjective fluorophotometry. Corneal epithelial dysfunction may renderthe cornea susceptible to a variety of pathologies, includingpotentially hazardous bacterial or fungal infections. Several systemicand ocular conditions are associated with reduced barrier function ofthe cornea, thus increasing vulnerability to the above complications. Anexample is the diabetic population, which presents a 5-fold decrease incorneal barrier function. Aging is also associated with reducedepithelial barrier function, with an exponential increase in epithelialpermeability with advanced age. A third example is the common conditionkeratoconjunctivitis sicca (keratitis sicca or dry eye), which causescorneal punctated epithelial lesions and increases permeation. Manytypes of eye drops have been developed to improve disturbed cornealepithelial surface but their efficiency is limited, and non-complianceis still a widespread problem. A possibility to decrease cornealpermeability could help millions of patients suffering from mild orsevere corneal barrier defects. It has been shown that exposure tomagnetic fields modulates vascular tone and permeability of some tissues[see, for example, Okano H. et al.: Bioelectromagnetics20(3)(1999)161-71]. It is therefore an object of the invention to reducethe corneal permeability by employing magnetic stimulation.

It is another object of the invention to provide a device for treatingeye disorders, comprising noninvasively reducing the cornealpermeability by the use of magnetic field.

It is also an object of the invention to provide a noninvasive magneticsystem for eye treatment by enhancing corneal barrier function.

Other objects and advantages of present invention will appear asdescription proceeds.

SUMMARY OF THE INVENTION

The invention provides a magnetic device for treating an eye or eyes,comprising a generator of a changing magnetic field having a strength offrom 0.1 to 5 mT in the vicinity of the eyes. Said magnetic field may bean alternating magnetic field of a frequency of from 5 Hz to 500 Hz. Themagnetic field in a device of the invention is preferably generated inpulses, the signal pulses having a length of from 1 to 10 second. Themagnetic field is preferably applied to the eyes in a repetitive manner;the field is created in the vicinity of an eye or eyes in repeatedsignal pulses. Two subsequent pulses generated by the device of theinvention are preferably separated by a time interval of from 1 to 10pulse lengths, and the total number of pulses generated by the device ofthe invention during a usual treatment procedure is from 10 to 100,providing the preferred total duration of the magnetic signal of from 10to 1000 seconds.

In a preferred embodiment of the invention, provided is a magneticdevice or system for treating the eyes, comprising a generator of achanging magnetic field, applied to said eyes or eye, having a strengthof from 0.1 to 5 mT in the vicinity of said eyes or eye, and a frequencyof from 5 Hz to 500 Hz, the field being generated in pulses having alength of from 1 to 10 second, such as 1 to 8 s, or 2 to 6 s, whereasthe total duration of the pulses, which is the product of the pulselength and the pulse number, is preferably from 10 to 1000 seconds, suchas 20 to 600 s, or 30 to 300 s, or 40 to 180 s, or 60 to 120 s. Amagnetic device for treating the eyes according to one embodiment of theinvention is configured to be worn by a subject in need of thetreatment. The device for treating the eyes according to the inventionhas in one embodiment a form of eyeglasses. The magnetic deviceaccording to the invention is advantageously employed in treating orpreventing conditions selected from the group consisting of eye dryness,keratitis sicca, corneal keratitis, corneal epithelial dysfunctions,reduced barrier function of the cornea associated with diabetes,conditions associated with increased corneal permeability due to ageing,minor lesions of the corneal surface, conditions associated with wearingcontact lenses, reduced self-healing capabilities of the cornea,penetration of harmful agents to the eye from the contaminatedenvironment, weakened anti-penetration system, and cornea-associatedinflammation.

The invention provides a method of noninvasively treating an eye of asubject, comprising creating a changing magnetic field in the vicinityof the cornea of said eye, the field having a strength of from 0.1 to 5mT, a frequency of from 5 Hz to 500 Hz, and pulse length of from 1 to 10second, thereby reducing the corneal permeability. The total duration ofthe pulses, which is the product of the pulse length and the pulsenumber, is preferably from 10 to 1000 seconds. In a preferred embodimentof the method according to the invention, corneal permeability isreduced by the magnetic field, whereby protecting the eye againstentrance of noxious agents or against loss of water. The inventionprovides a method of protecting the eye, comprising applying a changingmagnetic field to the eye of a subject in need of the treatment, wherebyreducing the corneal permeability. The method according to the inventioncomprises treating or preventing conditions selected from the groupconsisting of eye dryness, keratitis sicca, corneal keratitis, cornealepithelial dysfunctions, reduced barrier function of the corneaassociated with diabetes, conditions associated with increased cornealpermeability due to ageing, minor lesions of the corneal surface,conditions associated with wearing contact lenses, reduced self-healingcapabilities of the cornea, penetration of harmful agents to the eyefrom the contaminated environment, weakened anti-penetration system, andcornea-associated inflammation.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that certain types of the magnetic field reducethe permeability of the cornea. For example, changing magnetic field ofabout 0.9 mT substantially reduced the permeability of compromisedrabbit cornea toward fluorescein, for example when repetitively appliedin 3-second pulses interrupted by 15 second intervals, repeated 36times, at frequency of 20 Hz, one hour prior to permeability testing.

When relating to a changing magnetic field, any magnetic field whichdoes not have constant strength and direction is comprised, includingalternating or pulsed magnetic field. It is understood that changingmagnetic field comprises an electric component (electromagnetic field),but mainly the strength of the magnetic component is related to in thepresent invention.

The alternating magnetic field can induce electric fields in tissues [P.J. Maccabee et al.: J.Clin. Neurophysiol. 8(1) (1991) 38-55], it mayaffect cells growth, it may modulate vascular tone and permeability bymodulating calcium channels in vascular smooth muscle cells [Okano H. etal.: Bioelectromagnetics 20(3) (1999) 161-71]. Determination ofpotential mechanisms involved in this physiological responses tomagnetic field exposure is ongoing, and multiple and variable biologicalmechanisms have been suggested. Magnetic field has been also used inrepetitive trans-cranial magnetic stimulation, which is a process inwhich excitable human brain tissue is activated with an electric fieldinduced by a changing magnetic field, and the technology has been knownfor over 40 years as a reversible, localized, and noninvasive treatmentin various psychiatric disorders. Repetitive Magnetic Stimulation (rMS)is based on creating alternating magnetic fields by brief electriccurrents transmitted through an insulated coil. The present inventionaims at utilizing rMS electromagnetic pulses, similar as in repetitivetranscranial magnetic stimulation, for affecting the corneal tissue andreducing the corneal permeability.

The magnetic field employed in a system according to the presentinvention includes electromagnetic field of which magnetic component hasa strength of from 0.1 to 5 mT, such as from 0.1 to 2.5 mT, or from 0.1to 2 mT, or from 0.2 to 2.0 mT, for example around 1 mT. Said strengthis considered in the vicinity of the eye to be treated, which usuallyincludes the distance of from 0 and about 5 cm, such as from about 0.5to about 2.5 cm, similar to the distance of the eyeglasses from the eyesurface. The magnetic field may be an alternating field or pulsed fieldhaving a frequency between 5 to 500 Hz, such as 20 Hz. The field ispreferably applied repetitively, comprising signal pulses of between 1and 10 s, such as 3 s, separated by intervals having from 1 to 10 signalpulse lengths, such as 5, the total treatment time being usually between1 and 60 minutes, such as between 2 and 20 minutes, or between 3 and 15minutes, or between 4 and 12 minutes, for example up to about 20minutes, or up to about 15 minutes, or up to about 12 minutes, or up toabout 10 minutes, or up to about 6 minutes.

The invention provides, in one embodiment, a device that can be worn bya person, in which a magnetic field generator is incorporated. Thedevice may have a form similar to regular eyeglasses. The generator cangenerate a magnetic field of different parameters. The magnetic fieldreaches the eye. The decrease of the corneal permeability protects thecornea from physical damages, for example in persons suffering from eyedryness. In one embodiment of the invention, the magnetic field will begenerated by a special contact lens.

Certain magnetic stimulations decrease the permeability of the cornea,beneficially affecting persons suffering from eye dryness or personswearing contact-lenses, leading to decreased lubrication of the eye,known to make the cornea more susceptible to different types of damages.It has been found by the inventors that repetitive magnetic stimulationreduces the corneal permeability as desired. For example, changingmagnetic field which has a frequency of 5-500 Hz, such as 10-100 Hz, forexample 20 Hz, and which has an intensity of 0.1-5 mT, such as 0.2-4 mTor 0.3-3 mT, or 0.4-2 mT, for example about 1 mT, enhances the cornealintegrity and so protects the eye from damages eventually caused byundesired entry of damaging agents from the environment and/or undesiredexit of eye liquids.

This invention provides a device, a method, and a system for reducingthe corneal penetrability by applying magnetic field, assistingclinicians and pharmacologists challenged by eye disorders in which theentrance of noxious factors cannot be efficiently controlled. Theinvention enables to treat or to prevent or to mitigate the conditionsselected from the group consisting of eye dryness, keratitis sicca,corneal keratitis, corneal epithelial dysfunctions, reduced barrierfunction of the cornea associated with diabetes, conditions associatedwith increased corneal permeability due to ageing, minor lesions of thecorneal surface, conditions associated with wearing contact lenses,reduced self-healing capabilities of the cornea, penetration of harmfulagents to the eye from the contaminated environment, weakenedanti-penetration system, and cornea-associated inflammation. Theconditions to be handled by the device and method of the invention maybe associated with items selected from the group consisting of keratitiscaused be contact lenses, epidemic keratoconjunctivitis, aging of thecornea, epithelial corneal dystrophies, atopic keratoconjunctivitis,vernal keratoconjunctivitis, allograft corneal epithelial rejection,limbal chemical burn, epithelial keratitis—herpes simplex, epithelialkeratitis—neurotropic, Sjogren's syndrome, tear production induced byanti-Parkinson agents or anti-spasmotic agents or antiulcer-agents oraqueous tear deficiency medication, staphylococcal belephritis, argonlaser burns, Reiter syndrome, rheumatoid peripheral ulcerativekeratitis, and systemic diseases.

In exemplifying some embodiments of the invention, ocular penetration ofsodium-fluorescein in rabbit eyes following magnetic stimulation atdifferent intensities was used. The model using rabbit eyes and sodiumfluorescein was employed by the inventors due to the high ocular safetyprofile of the compound, its hydrophilic nature, and the fact that itsconcentration in the anterior chamber can be measured with goodprecision and reproducibility using a fluorometer. The baseline cornealpermeability of this hydrophilic substance is very limited, due to thecorneal barrier function. It is known that fluorescein does notpenetrate or stain live corneal epithelial cells upon topicalapplication, the corneal epithelial defects are readily stained as thedye diffuses between cells into the adjacent intercellular spaces andpenetrates into the underlying corneal stroma. Rabbits were either nontreated (sham) or treated with repeated magnetic stimulation (rMS) atdifferent intensities one hour prior to fluorescein application.Fluorescence measurements of anterior chamber fluid was used to quantifythe ocular penetration. In order to demonstrate the safety of theprocedure, animals were tested for retinal function byelectro-retinogram prior to rMS treatment, at 1 day, 1 week, and 1 monthfollowing the treatment. Moreover, animals were examined by histologicalanalysis for possible adverse effects on retinal structure.

The invention enables to enhance the corneal integrity. Ocular surfacedisease comprises numerous disorders affecting millions around theworld, and is a problem encountered routinely in daily practice. Asubgroup of these patients suffers from a chronic compromise of thecorneal surface, which in turn may result in corneal scarring,infection, thinning and ultimately perforation. In this subgroup, theself healing capabilities of the cornea are significantly impaired incomparison to normal corneas. A method for enhancing corneal integrityand reducing permeability may serve to help protect these compromisedcorneas, and may even facilitate accelerated healing. The inventionenables to reduce the permeability in cornea subjected to magneticstimulation, so safeguarding the ocular surface in these delicatesituations. To gain insight on the mechanisms underlying rMS cornealeffects, the possibility that rMS enhances epithelial recovery waschecked, including monitoring the corneal erosion area in sham and rMStreated animals. Without wishing to be bound by any theory, theinventors assume that the alternating magnetic field which was employedacts both directly and also through the induced electrical field,affecting both ions and dipoles on the molecular level, as well as thewhole cells.

The invention provides a magnetic system and device comprising agenerator of changing magnetic field, enabling repetitive magneticstimulation, having a strength of from 0.1 mT to 5 mT and a frequency offrom 5 Hz to 500 Hz, the field generated in pulses having a length offrom 1 to 20 second, separated by intervals of from 1 to 30 second. Theterm “strength” in regard to the magnetic field is used in the samesense as the term “intensity”, intending the field magnitude measured inthe units of tesla (T). The system and device of the invention employ,in some embodiments, magnetic field of 0.1-10 mT, for example 0.1-5 mT,or 0.2-4 mT, or 0.3-3 mT, or 0.5-2 mT. The system and device of theinvention employ, in some embodiments, alternating magnetic field of5-500 Hz, for example 10-250 Hz, or 10-100 Hz, or 10-50 Hz. The systemand device of the invention employ, in some embodiments, alternatingmagnetic field in pulses having duration 1-100 s, for example 1-20 s, or2-20 s, or 2-10 s. The system and device of the invention employ, insome embodiments, alternating magnetic field in pulses separated by timeintervals without signal of the same length as the pulse duration ormore, for example an interval twice as long as the pulse duration, orthree times as long as the pulse duration, or four times as long as thepulse duration, or five times as long as the pulse duration, or from sixtimes to ten times as long as the pulse duration.

The system and device of the invention enable a noninvasive eyetreatment by enhancing the corneal barrier function.

In a preferred embodiment, the system and device of the invention assistin protecting the cornea by maintaining normal hydration and retainingocular surface integrity via reducing the corneal permeability.

The invention will be further described and illustrated in the followingexamples.

EXAMPLES

Materials and Methods

Animals—Twenty New Zealand white rabbits, an outbred stock used indermatological, ophthalmological and other areas of biomedical research.Animals were housed at the Glodschlager Eye Research Institute animalfacility.

Compromised corneal epithelium was achieved by anesthetizing animalswith xylazine-ketamine and keeping their eyes open for 90 minutes.Clinical signs of dry eye can be observed after this time period in theform of acute desiccation.

Dye application and anterior chamber tap—5 microliter of 10% sodiumfluorescein was instilled on rabbit corneas pre-evaluated for theabsence of epithelial damage using a slit-lamp. After 30 seconds ofresidence time on the ocular surface, the eye was washed out thoroughlyusing normal saline for 5-7 minutes or more as needed. Taps (100 μl)were taken from the anterior chambers of the rabbit eyes atpredetermined intervals during 60 minutes after instillation of thefluorescein. For rose bengal staining, 0.5% solution was used.

Fluorometry measurement—using a fluorometer (FL×800 FluorescenceMicroplate Reader, BioTek) the anterior chamber fluid was analyzed forfluorescein concentration at wavelengths of 485/20 (excitation) and528/20 (emission).

Magnetic stimulation—rMS (repeated magnetic stimulation) was applied toone eye pre-evaluated for the absence of epithelial damage. The eye wasexposed to single session of rMS at varying output intensities (0 to 1mT), 20 Hz stimulus for 3 seconds, followed by a 15 second interval withno stimulus (no signal). This was repeated 36 times. Following the aboverepetitive stimulation, fluorescein was instilled and anterior chamberfluid analyzed.

Electroretinogram (ERG)—Retinal function of all the animals was testedprior to rMS treatment, and at one day, one week and one month aftertreatment. Animals was dark-adapted for a minimum of 6 hours prior toERG measurements. Animals were anesthetized with intraperitonealKetamine and Xylazine. Pupils were dilated with topical 1% tropicamide.The corneas were kept moist with 2.5% hydroxypropyl methylcellulose.Body temperature were kept at 37° C. with a heating pad. ERGs wererecorded from both eyes simultaneously using golden wire loops on thecorneas, in five increasing intensities (−20, −10, 0, +2.5, +10 dB). Achloride silver reference electrode was placed subcutaneously near thetemporal eye corner. The ground electrode was placed on the tail.Scotopic ERG responses was averaged with stimulus intervals of 1 to 30seconds depending on intensity, and 20 photopic responses were averagedwith stimulus intervals of 1 second. Animals was light-adapted for 5minutes before photopic recordings.

Histology—Two rabbits were sacrificed and their eyes fixed in 3.7%formaldehyde for 24 hrs and embedded in paraffin. Paraffin sections werestained with hematoxylin and eosin, and examined for eye morphology.

Example 1

500 μg sodium fluorescein was introduced onto rabbit corneas with intactor compromised epithelia, followed by withdrawing anterior chamber tapsat different time intervals after washout. A cornea with a compromisedepithelium was defined as one with any sign of staining immediatelyafter initial washout of the dye. The erosions seen in our preliminarystudy varied slightly in size (⅕-⅓ of corneal surface); however, nosignificant variations in permeability were noted. The fluoresceinconcentration in the anterior chamber increased with time, with a peakat 60 min post application. Moreover, fluorescein concentration in theanterior chamber was significantly higher, by up to 100 fold, in eyeswith compromised epithelium. The concentration of fluorescein found inthe anterior chamber during the different time intervals after washoutpresented similar dynamics to those found in a study on human eyescomparing normal to patients with corneal damage and increasedepithelial permeability [Matsuo H. et al.: Am. J. Ophthalmol. 140(4)2005) 742-4].

The effects of magnetic stimulation on corneal penetration were checkedin rabbits treated in one eye with rMS at 940 μT, 20 Hz, 36 repeats 1 hrprior to fluorescein application. The rabbit Motor Threshold was at 890μT (microtesla). Hence an intensity higher than the motor threshold waschosen. Magnetic stimulation at 940 μT significantly reduced fluoresceinpenetration in eyes with compromised epithelium to a level similar tothat obtained in eyes with intact epithelium. These findings demonstratethat rMS treatment can minimize barrier defects arising from epithelialerosion, suggesting that rMS may be used for treatment in patients withcompromised corneal epithelium.

Example 2

500 μg sodium fluorescein was introduced onto rabbit corneas with intactor compromised epithelia, followed by withdrawing anterior chamber tapsat different time intervals after washout. Rabbits, either sham or rMStreated at different intensities, 1 h prior to fluorescein applicationwere divided to 4 groups (N=5 in each): Group A received sham treatment;groups B, C and D magnetic stimulation at 760, 890 and 940 microtesla,respectively. The fluorescence measurements values were averaged in eachgroup. The fluorescence average values in groups B, C, and D were about110%, 50%, and 20%, respectively, relatively to A.

The effect of rMS on corneal damage was histopathologically examined.Corneal specimens were stained with hemotoxylin and eosin. Innon-treated dry eye, local erosion of several epithelial cell layers wasobserved, whereas the histological structures of the posterior stromalcell layer and the basement membrane remained intact. By contrast, ineyes pretreated with rMS, corneas were demonstrated to have the stromalcell layer uniformly overlaid with intact multilayered epithelium.

Further, to evaluate how long the therapeutic effect of rMS lasts, threerabbits were treated with rMS on one eye as indicated above. Three weeksafter a single rMS session, dry eye was induced in both eyes therabbits, and fluorescein concentration in the anterior chambers tap weremeasured 60 minutes after application of fluorescein. Significantlyreduced fluorescein penetration was observed in the treated eyes,compared to the non-treated ones.

To evaluate safety of rMS treatment, fifteen rabbits were monitored forup to six months following rMS treatment. No adverse effects on generalhealth, body weight, or cataract development were found in any animal.In addition, eyes removed five hours and 3 months, respectively, afterrMS treatment were analyzed by histopathology, and no adverse effectswere found in any eye; specifically retinal structures wereundistinguishable in the two eyes and in untreated control eyes, whenchecked on paraffin sections stained with hematoxylin and eosin, or withGiemsa stain.

The findings demonstrated that the rMS treatment according to theinvention can minimize barrier defects arising from dry eye for at leastthree weeks in mammals, with no adverse effects, suggesting that rMS maybe advantageously used for treating patients suffering from dry eye.

While this invention has been described in terms of some specificexamples, many modifications and variations are possible. It istherefore understood that within the scope of the appended claims, theinvention may be realized otherwise than as specifically described.

The invention claimed is:
 1. A method of treating an eye of a subject,comprising i) applying a changing magnetic field; ii) measuringpenetration of fluorescein from the corneal surface to anterior chamberof said eye; and iii) selecting a magnetic field strength sufficient toreduce corneal permeability.
 2. The method of claim 1, wherein thecorneal permeability is reduced by the magnetic field, therebyprotecting the eye against entrance of noxious agents or against loss ofwater.
 3. The method of claim 1, comprising repetitively applying saidmagnetic field to the eye of said subject in need of said treating,thereby reducing the corneal permeability.
 4. The method of claim 1,comprising treating or preventing conditions selected from the groupconsisting of keratitis sicca, eye dryness, corneal epithelialdysfunctions, corneal keratitis, reduced barrier function of the corneaassociated with diabetes, conditions associated with increased cornealpermeability due to ageing, minor lesions of the corneal surface,conditions associated with wearing contact lenses, reduced self-healingcapabilities of the cornea, and penetration of harmful agents to the eyefrom a contaminated environment.
 5. The method of claim 1, wherein saidstrength is sufficient to reduce the corneal permeability as measured bythe penetration of fluorescein from the corneal surface to the anteriorchamber.
 6. The method of claim 1, comprising treating eye dryness.